| Objective To generate humanizedβ-thalassemia mice models and construct recombinant lentiviral(LV) vectors carrying humanα-antisense oligonucleotide, to explore the role of lentiviral vectors carrying humanα-antisense oligonucleotide in humanizedβ-thalassemia mice models.Methods 1.The human bone marrow or cord blood mononuclear cells were transplanted into NOD/SCID mice pretreated with the different schemes and dosage. After transplantation, human recombinant growth factors consisting of rhEPO and rhGM-CSF were injected into the mice intraperitoneally. The survival status of the mice was recorded to calculate death rate. Six weeks after the transplantation, flow cytometric detection of human cells in murine tissues (bone marrow and blood) was performed with monoclonal antibodies against human CD45.2.The bone marrow mononuclear cells ofβ-thalassemia patients were transplanted into the sublethally irradiated NOD/SCID mice. After transplantation, human recombinant growth factors consisting of rhEPO and rhGM-CSF were injected into the mice intraperitoneally. Five weeks after the transplantation, flow cytometric detection of human cells in murine tissues (bone marrow and blood) was performed with monoclonal antibodies against human CD45. Electrophoresis of hemoglobin, analysis of hemoglobin chains and detection ofβ-thalassemia gene mutations were performed in murine tissues (bone marrow or blood). Theα-globin chain precipitates in erythroid cells were observed in electron microscopy.In addition,spleen and liver of the mice were removed, observed,embedded,and sectioned.Specimens were stained with hematoxylin and eosin and iron stains (Prussian blue).3.According to the humanα-antisense oligonucleotide sequence, pGCSIL- vshRNA-GFP plasmid was constructed by double restriction enzyme digestion and ligation, and then the plasmid was transformed into E.coliDH5α. Purified pGCSIL-vshRNA-GFP plasmids from the positive clones was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector pGCSIL-vshRNA-GFP, pHelper 1.0 and pHelper 2.0 by Lipofectamine 2000 to produce lentivirus. The titer of virus was tested according to the expression level of green fluorescent protein. In addition, with the same methods, recombinant lentiviral vectors for NC sequence were constructed.4.The bone marrow CD34+ cells ofβ-thalassemia patients were transduced by lentiviral vectors carrying humanα-antisense oligonucleotide. Then the transduced cells were transplanted into the sublethally irradiated NOD/ SCID mice by tail vein injection,while non- transducedβ-thalassemia cells,β-thalassemia cells transduced by NC-GFP-LV and normal bone marrow cells were used as controls. After transplantation, the general conditions such as body weight of the mice were observed. Six weeks after the transplantation, flow cytometric detection of human cells in murine tissues (bone marrow and blood) was performed with monoclonal antibodies against human CD45. Electrophoresis of hemoglobin and detection ofβ-thalassemia gene mutations were performed in murine tissues (bone marrow or blood). In addition, spleen and liver of the mice were removed, observed, embedded, and sectioned. Specimens were stained with hematoxylin and eosin and iron stains. Results 1.The death rates of the mice in TBI/CY bone marrow transplantation group and TBI/CY cord blood transplantation group were all 100%.While the death rates of the mice in TBI bone marrow transplantation group and TBI cord blood transplantation group were all 33.3%. In TBI cord blood transplantation group, the percentage of human CD45+ cells in the bone marrow of the No. 13 and the No.6 mouse was 59.61% and 21.46%, respectively at week 6 after transplantation. While in TBI bone marrow transplantation group and control group, the percentage of that of each mouse was 0, respectively.2.Five weeks after transplantation, in theβ-thalassemia/HbE bone marrow transplantation group, the percentages of human CD45+ cells in murine tissues (bone marrow and blood) were 7.21% and 4.08% in +47 day, 7.37% and 6.03% in +61 day,respectively. And in theβ-thalassemia bone marrow transplantation group , those were 8.17% and 3.43% in +36 day, 16.82% and 8.89% in +45 day,respectively. There were HbA2 bands in hemoglobin electrophoresis of the transplantation group mice. Theirα-globin chain bands were thicker and wider than theβ-chain in hemoglobin chains analysis. In the transplantation group mice, the results ofβ-thalassemia gene mutations correspond with the donor. There wereα-globin chain precipitates in some erythroid cells.The ratios of the spleen length to body weight of the transplantation group mice were greater than those of control group[(0.5762±0.0451)vs(0.3628±0.0340)mm/g,P=0.000]. The iron deposition was observed in liver and spleen of the transplantation group mice.3.The exogenous gene sequence of the recombinant plasmids was completely in accordance with that of the humanα-antisense oligonucleotide. The titer of concentrated virus was 5×109TU/ml.4.Six weeks after the transplantation, the mice transplanted with normal bone marrow cells and blank mice became heavier, and other mice lost or maintained their weight. In bone marrow and blood of the mice being transplanted with human bone marrow cells, human CD45+ cells could be detected. In mice being transplanted withβ-thalassemia bone marrow cells (non-transduced or transduced by LV), the results ofβ-thalassemia gene mutations correspond with the donor. The ratio of the spleen length to body weight of the mice being transplanted withβ-thalassemia bone marrow cells (non-transduced or transduced by LV) were greater than that of other mice([0.6848±0.0886)v(s0.5573±0.0088)mm/g,P=0.016]. The iron deposition observed in liver and spleen of the mice being transplanted withα-ASON- LV-transducedβ-thalassemia bone marrow cells was less than that of the mice transplanted with non-transduced or NC-GFP-LV-transducedβ-thalassemia bone marrow cells.Conclusions The method whichβ-thalassemia bone marrow cells were transplanted into the sublethally irradiated NOD/SCID mice can generate humanizedβ-thalassemia mice models succesfully. The iron deposition of humanizedβ-thalassemia mice models was possibly reduced by lentiviral vectors carrying humanα-antisense oligonucleotide. The role of lentiviral vectors carrying humanα-antisense oligonucleotide in humanizedβ-thalassemia mice need to be observed continually.
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